Research Summary Human cytomegalovirus (CMV) is a ?-herpes virus that increases morbidity and mortality in immuno- compromised individuals. The major populations at high risk for CMV-associated diseases are unborn children and transplant recipients. Currently, CMV disease is treated with anti-viral drugs including ganciclovir (GCV), valganciclovir (valGCV), foscarnet (PFA), and cidofovir (CDV). Despite the efficacy of these drugs, there are severe limitations that include poor oral bioavailability, dose-related toxicity, and development of drug resistant viral mutants. Moreover they are precluded for use in pregnant women. An alternative therapy is human anti- cytomegalovirus immune globulin (CMV-IGIV, Cytogam) derived from pooled adult human plasma that shows limited effectiveness. The numerous shortcomings of the current therapies and the lack of pharmaceutical options to prevent and treat CMV disease warrant the development of more potent and safer anti-CMV therapies. Thus, our objective of this R21 grant is to develop antibody-drug conjugates (ADC) using antibodies that recognize viral envelope proteins that target virus-infected cells. During virus infection, the envelope proteins gB and gH are expressed on the cell surface during the late phase of virus replication. The surface expressed envelope proteins represent biomarkers for virus-infected cells that could be specifically targeted by biologics to limit virus proliferation. The specificity of an ADC would selectively inhibit virus dissemination in cells actively replicating virus. We hypothesize that targeting the membrane envelope proteins gB and gH of virus-infected cells with ADCs will effectively prevent virus propagation and dissemination. Utilizing CMV neutralizing monoclonal antibodies (mAbs) against envelope proteins gB and gH, we plan to accomplish our objective by: 1) Characterizing the anti-CMV mAbs that broadly recognize virus-infected cells. Monoclonal antibodies (mAbs) directed against CMV envelope proteins to diverse epitopes of gB and gH will be labeled with a fluorophore to characterize the mAbs that efficiently bind to virus infected cells and are internalized during the virus life cycle. And 2) Determining the efficacy of ADCs to limit viral replication and dissemination. The anti-gB and -gH mAbs with the highest level of internalization in virus-infected cells will be conjugated with the antiviral drug ganciclovir or an antimitotic agent (a new-generation toxoid) to determine conditions that effectively limit virus proliferation in multi-cycle growth assays. We are expecting to identify the mAbs that are amenable to drug conjugation to be utilized as potential ADCs to limit CMV replication and dissemination.